Daniel:Notebook/ComboLock/2016-10-12

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Padlock0301 Test (Started Friday 10-7)[edit]

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Testing the amplification using the forward (AmpF-CL-v2) and primer24 reverse set, as well as some dilutions.

Protocol[edit]

  1. qPCR
    1. Make the following master mixes
    2. Master Mix nfH20 AmpF-CL-v2 Reverse Primer Rev. Primer Conc. (uM) Amt. Reverse Primer Kapa SYBR Fast MM Total
      MM1 90.2 2.05 primer24 100 2.05 102.5 196.8
      MM2 93.48 0.41 AmpR6.3-27 100 0.41 102.5 196.8
      MM3 90.2 2.05 AmpR6.3-28 10 2.05 102.5 196.8
      MM4 93.48 0.41 AmpR6.3-29 10 0.41 102.5 196.8


    3. Set up qPCR reactions according to table in next section
    4. Aliquot 48 uL correct master mix into each well
    5. Add 2 uL sample to appropriate well
    6. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x26
      7. 72C 2 min
      8. 16C hold
  2. TBE Gel
    1. Mix 48 uL TBE, 12 uL 6x loading dye
    2. Aliquot 10 uL per sample/ladder lane onto parafilm
    3. Add 2 uL of sample or ladder to correct drop
    4. Load 10 uL in to well
    5. Run gel for 22 minutes at 250V
    6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc




qPCR Table[edit]

Sample Molecule Count Lane Reverse Primer Reverse Primer Conc. (uM) Sample Vol (uL) 2X Kapa SYBR qPCR MM Reverse Primer 100 uM AmpF H2O Total Volume (uL) Master Mix
Sample 0A 10 pmol A1 primer24 100 2 25 0.5 0.5 22 50 MM1
Sample 0B 10 pmol A2 primer24 100 2 25 0.5 0.5 22 50 MM1
Sample 0C 10 pmol A3 primer24 100 2 25 0.5 0.5 22 50 MM1
NTC (rev) 0 A4 primer24 100 0 25 0.5 0.5 24 50 MM1
Sample 0A 10 pmol A1 AmpR6.3-27 100 2 25 0.1 0.1 22.8 50 MM2
Sample 0B 10 pmol A2 AmpR6.3-27 100 2 25 0.1 0.1 22.8 50 MM2
Sample 0C 10 pmol A3 AmpR6.3-27 100 2 25 0.1 0.1 22.8 50 MM2
NTC (rev) 0 A4 AmpR6.3-27 100 0 25 0.1 0.1 24 50 MM2
Sample 0A 10 pmol A1 AmpR6.3-28 10 2 25 0.5 0.5 22 50 MM3
Sample 0B 10 pmol A2 AmpR6.3-28 10 2 25 0.5 0.5 22 50 MM3
Sample 0C 10 pmol A3 AmpR6.3-28 10 2 25 0.5 0.5 22 50 MM3
NTC (rev) 0 A4 AmpR6.3-28 10 0 25 0.5 0.5 22 50 MM3
Sample 0A 10 pmol A1 AmpR6.3-29 10 2 25 0.1 0.1 22.8 50 MM4
Sample 0B 10 pmol A2 AmpR6.3-29 10 2 25 0.1 0.1 22.8 50 MM4
Sample 0C 10 pmol A3 AmpR6.3-29 10 2 25 0.1 0.1 22.8 50 MM4
NTC (rev) 0 A4 AmpR6.3-29 10 0 25 0.1 0.1 24 50 MM4

Results[edit]

Library Prep[edit]

  1. qPCR
    1. Make the following master mix
      1. 71.75 uL nfH2O
      2. 2.05 uL AmpF-CL-6.4
      3. 102.5 uL Kapa SYBR Fast
    2. Aliquot 43 uL master mix into each well
    3. Prep samples according to following table
    4. Sample Template Molecule Count Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 100 uM AmpF-CL-v2 10 uM AmpR6.3-IndXX H2O Total Volume (uL)
      Sample 1A 10 pmol A1 24 2 25 0.5 5 17.5 50
      Sample 1B 10 pmol B1 24 2 25 0.5 5 17.5 50
      Sample 1C 10 pmol C1 24 2 25 0.5 5 17.5 50
      NTC 0 D1 25 0 25 0.5 5 19.5 50
    5. Add 2 uL sample to appropriate well
    6. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x26
      7. 72C 2 min
      8. 16C hold

    File:20161012-qpCR-Padlock0301-LibraryPrep.png

  2. Size Select Gel
    1. Mix 20 uL sample and 4 uL 6x loading dye into separate tubes for each sample
    2. Mix 4 uL ladder, 4 uL 6x dye, and 16 uL TBE in ladder tube
    3. Aliquot 24 uL per sample/ladder lane onto parafilm
    4. Add 2 uL of sample or ladder to correct drop
    5. Load 10 uL in to well
    6. Run gel for 22 minutes at 250V
    7. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    8. Rinse gel and image in gel doc
    9. Cut out appropriate bands with scalpel; put in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube
    10. Image in gel doc
    11. Centrifuge at 12000 rpm for 1.5 minutes
    12. Add 500 uL TE to each sample
    13. Incubate overnight with vigorous shaking; continued tomorrow